Arginine Deprivation in SCLC: Mechanisms and Perspectives for Therapy

Abstract Arginine deprivation has gained increasing traction as a novel and safe antimetabolite strategy for the treatment of several hard-to-treat cancers characterised by a critical dependency on arginine. Small cell lung cancer (SCLC) displays marked arginine auxotrophy due to inactivation of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1), and as a consequence may be targeted with pegylated arginine deiminase or ADI-PEG20 (pegargiminase) and human recombinant pegylated arginases (rhArgPEG, BCT-100 and pegzilarginase). Although preclinical studies reveal that ASS1-deficient SCLC cell lines are highly sensitive to arginine-degrading enzymes, there is a clear disconnect with the clinic with minimal activity seen to date that may be due in part to patient selection. Recent studies have explored resistance mechanisms to arginine depletion focusing on tumor adaptation, such as ASS1 re-expression and autophagy, stromal cell inputs including macrophage infiltration, and tumor heterogeneity. Here, we explore how arginine deprivation may be combined strategically with novel agents to improve SCLC management by modulating resistance and increasing the efficacy of existing agents. Moreover, recent work has identified an intriguing role for targeting arginine in combination with PD-1/PD-L1 immune checkpoint inhibitors and clinical trials are in progress. Thus, future studies of arginine-depleting agents with chemoimmunotherapy, the current standard of care for SCLC, may lead to enhanced disease control and much needed improvements in long-term survival for patients.

Introduction
Small cell lung cancer (SCLC) is a notoriously lethal subtype of smoking-related thoracic cancer, accounting for 10-15% of global lung cancer incidence with a 5-year survival rate of less than 7%. 1 Treatments for SCLC have advanced slowly in the last few decades with much scope for improvement. Harnessing fundamental and novel biological insights will be critical to transforming the SCLC therapeutic landscape.
Derived from neuroendocrine precursor cells, SCLC is characterised by rapid tumor growth, genomic instability and early metastatic spread underlying the poor survival outcomes. 2,3 On a molecular level, in addition to universal TP53 and RB1 inactivation, SCLC is classified into four distinct subtypes according to the expression of ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) or YAP1 (SCLC-Y). 4,5 This molecular reappraisal of SCLC offers the possibility of novel personalised therapies. Inducing arginine deprivation is a promising therapeutic strategy for multiple cancer types including SCLC that are deficient in the arginine biosynthetic enzyme argininosuccinate synthetase 1 (ASS1). In the current review, we provide an update on arginine deprivation as a specific antimetabolite therapy focusing on SCLC both as monotherapy and in combination with selected therapies based on emerging preclinical and clinical data.

Critical Use of Arginine in Healthy and Cancerous Cells
Background Arginine (2-amino-5-guanidinovaleric acid) is a semi-essential amino acid in mammalian cells. 6 As one of the 20 most common amino acids in humans, it is a highly versatile molecule serving as the precursor of key molecules such as urea, nitric oxide, proline, polyamines or proteins. 7 Additionally, arginine can trigger secretion of molecules such as growth hormones, insulin or glucagon. 8 This metabolite is a core component of the urea cycle, the only known pathway in humans capable of recycling and eliminating the nitrogen-containing molecule ammonia. 9,10 Arginine biosynthesis takes place within the urea cycle from the conversion of ornithine to citrulline in the mitochondria catalysed by the ornithine transcarbamylase (OTC) enzyme. 11 Citrulline can also be sourced from glutamine through a series of sequential intestinal enzymatic reactions which release the metabolite to the blood circulation. 12 Citrulline and aspartate undergo condensation to argininosuccinate by the ATP-dependent enzyme ASS1, the rate-limiting enzyme of the urea cycle. The last step is the conversion of argininosuccinate into arginine and fumarate, a member of the TCA cycle, by argininosuccinate lyase (ASL). 8 Arginine biosynthesis accounts for only 10-15% of whole-body arginine production, implying that cells rely primarily on the transport of extracellular arginine and therefore the cell microenvironment to maintain arginine levels. 13

ASS1 Modulation in Cancer
ASS1 inactivation confers arginine auxotrophy in cancer cells and thereby susceptibility to arginine-depleting agents. 14 Loss of ASS1 and sensitivity to arginine deprivation has been observed in numerous cancers including melanoma, hepatocellular carcinoma (HCC), malignant pleural mesothelioma, glioblastoma, sarcomas and haematological cancers. [15][16][17][18][19] Other malignancies, such as ovarian and colorectal cancer express high levels of ASS1 compared to corresponding normal tissues. 20 In SCLC, Kelly et al identified that 45% of tumor samples and 50% of cell lines were ASS1 negative highlighting the importance of arginine biosynthesis downregulation for this malignancy. 21 The mechanisms leading to altered ASS1 expression differ. Transcription factors such as c-Myc and HIF-1a interacting with an E-box located at the ASS1 gene promoter are known to modulate the expression of ASS. 22 ASS1 expression is also under epigenetic regulation with promoter methylation first being identified in mesothelioma cell lines and primary tumors as a driver of ASS1 deficiency. 16,23 Moreover, ADI-PEG20 treatment induced ASS1 re-expression in an MPM cell line linked to decreased methylation of the ASS1 promoter. 24 Although the exact mechanism of ASS1 expression in SCLC is yet to be fully understood, Chalishazar et al showed that MYC directly affects the arginine dependency of SCLC cell lines. 25 As such, MYC-driven cells were more sensitive to arginine deprivation, the reverse of that described for melanoma and highlighting cell-of-origin as a key determinant of the arginine metabolome. 8,26 Several studies have highlighted the functional and metabolic changes as a consequence of ASS1 dysregulation in cancer (Table 1). To summarise, ASS1 downregulation correlates frequently with more aggressive and chemoresistant cancer phenotypes including increased proliferation, migration, invasion, and metabolic changes affecting glycolysis and the TCA cycle. 27 Low levels of ASS1 were associated with worse overall survival for several cancer types including ovarian cancer, mesothelioma, non-small cell lung cancer, bladder cancer and osteosarcoma. [28][29][30][31][32] On the other hand, in gastric cancer, ASS1 expression has been linked with tumorigenesis, chemoresistance and poor outcomes. 33 Targeting Arginine for Cancer Treatment A deeper understanding of the role of arginine and ASS1 in tumorigenesis has accelerated the exploitation of arginine auxotrophy for personalised anticancer therapy. Several enzyme therapeutics have been developed to have a synthetically lethal effect in arginine-dependent and urea cycle-dysregulated tumor cells. 34

Arginase (ARG)
ARG is an enzyme that catalyses the conversion of arginine into ornithine and urea. Recombinant human ARGI has been conjugated with polyethylene glycol of 5kDa to increase its half-life creating the clinically usable molecule: rhArg-PEG (BCT-100). Xu et al evaluated the potency of rhArg-PEG (BCT-100) on several SCLC cell lines, revealing apoptosis induced by oxidative stress, G1 cell cycle arrest, and reduced arginine concentrations correlating with low levels of ASS1 and/or OTC. 35 rhArg-PEG (BCT-100) has been studied in several phase I and II clinical trials in patients with HCC, melanoma, prostate adenocarcinoma or leukaemia; however, no data are available specifically in SCLC. [36][37][38][39] Another rhArgPEG that is cobalt-substituted (Co-Arg1-PEG or pegzilarginase) also displayed robust activity in 8/12 patientderived xenograft (PDx) models of SCLC and the related Merkel cell cancer, an aggressive skin neuroendocrine cancer.

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There was a good correlation between SCLC PDx sensitivity to pegzilarginase (4mg/kg) monotherapy and negative or low ASS1 expression by immunohistochemistry, consistent with multiple cancer cell lines studies. A clinical trial of pegzilarginase in a dose-expansion cohort of patients with SCLC has completed accrual and results are awaited (ClinicalTrials.gov Identifier: NCT03371979).

Pegylated Arginine Deiminase
Arginine Deiminase (ADI) is an enzyme that can be found in several organisms (Streptococcus faecalis, Pseudomonas putida, Mycoplasma hominis, Mycoplasma arginini). This enzyme catalyzes the conversion of arginine into citrulline and ammonia. ADI has been formulated with polyethylene glycol (20kDa) to optimise the circulating half-life of the drug known as ADI-PEG20 (pegargiminase). In 2002, Ensor et al published the first study linking ASS1 expression to the efficacy of ADI-PEG20 in melanoma and liver cancer cell lines, leading to early phase clinical trials and FDA designation of ADI-PEG20 as an orphan drug for the treatment for malignant melanoma and HCC. 15,41,42 ADI-PEG20 is also cytotoxic to SCLC cell lines in vitro inducing caspase-independent cell death and autophagy. SCLC xenografts were also sensitive to ADI-PEG20 (5IU) with both small and established tumors responding in an ASS1-dependent fashion on par with that seen in SCLC PDx models with pegzilarginase. 21 Based on the promising preclinical data, a Phase II two-arm, non-randomised study of pegargiminase enrolled 22 patients with histologically confirmed SCLC in which <5% of tumor cells expressed ASS1 by immunohistochemistry. 43 Cohort 1 enrolled 9 patients with "sensitive" disease who had disease control for at least 90 days after 1 previous line of

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chemotherapy. Cohort 2 enrolled 13 patients with "refractory" disease who had either progressed while on chemotherapy or within 90 days of completing treatment. Both cohorts received treatment with weekly intramuscular ADI-PEG20 (320IU/m 2 or 36.8mg/m 2 ) and the primary endpoint for clinical efficacy was tumor response, defined as complete response or partial response by RECIST 1.1 criteria. Although, the study was terminated early as there was poor recruitment to the sensitive disease cohort and no radiologic responses to pegargiminase monotherapy, activity was identified in the refractory cohort with a patient experiencing temporary disease stabilisation and normalisation of serum LDH, a known prognostic and predictive factor in SCLC. 44 Sensitivity and Resistance to Arginine-Depleting Agents The effects of arginine deprivation on tumor cell molecular mechanisms and the metabolome are under active investigation and vary according to cell type. Much of the mechanistic data derives from studies of ADI-PEG20 and is discussed in further detail below and in Table 2. Broadly, a number of outcomes have been reported as follows: cell cycle arrest, caspase activation, PARP cleavage, autophagy, nucleotide and polyamine metabolism rewiring, and modulation of RAS-ERK and PI3K-mTOR activity. 45 In SCLC, much remains to be elucidated; however, several areas are of specific interest relating to the products of arginine catabolism. First, nitric oxide synthase (NOS) converts arginine into NO, a small reactive molecule with pleiotropic effects in host and cancer cells. 46 Indeed, NOS has a dominant role along with NOTCH in activating downstream soluble guanylate cyclase, a key pathway of platinum resistance in SCLC. Pharmacological depletion of NO via NOS blockade resensitized SCLC to chemotherapy. 47 NO also possesses key innate and adaptive immune signalling properties, with myeloid cell arginase -a known immunosuppressive pathway -directly competing with NOS for arginine. 48,49 Furthermore, inhibiting myeloid arginase and NOS has been shown to enhance tumor immunity in a variety of cancer models. 50 Interestingly, bronchoalveolar-derived leucocytes from patients with SCLC produce 6-fold greater levels of NO than those from patients with NSCLC. 51 Also, arginase-1 (ARG1) and ARG2 are expressed in SCLC in TAMs and tumor cells, respectively, with higher levels of ARG2 when compared to NSCLC, reinforcing the functional and clinical analysis of SCLC which is dependent on downstream polyamine biosynthesis. 25,[52][53][54] Apropos drug resistance, while most patients develop anti-ADI-PEG20 antibodies compared to a minority of patients developing anti-pegzilarginase antibodies, complete responses were nonetheless reported for pegargiminase but none for pegzilarginase in patients with acute myeloid leukaemia. 43,55,56 Clearly, tumor-intrinsic rather than drug-innate mechanisms of resistance are operational with emerging rebiopsy data from several phase I/II studies providing pertinent clues. Thus, one-third of patients with the thoracic cancers mesothelioma and NSCLC progressing on pegargiminase display tumoral ASS1 re-expression. 30,57 This is consistent with data reported using primary melanoma cell lines and also evident in patients with melanoma progressing on ADI-PEG20. 58,59 When ASS1 is re-expressed, cancer cells no longer depend on exogenous arginine and instead upregulate the urea cycle using citrulline generated by ADI-PEG20. Recent work by Xu et al identified contactin 1 as a mediator of rhArg-PEG (BCT-100) resistance in SCLC cell lines by promoting epithelial-mesenchymal transition linked to upregulation of AKT. 60 In addition to specific alterations within tumor cells -and, notwithstanding the known limitations of obtaining rebiopsy tissue in SCLC -a critical component of resistance may be mediated by the stromal compartment. Thus, we showed ASS1-expressing macrophages highly upregulated in relapsing thoracic cancers linked to a pro-tumor chemokine network. 57 The role of macrophages in SCLC is being leveraged with the development of CD47 antibodies and this may provide opportunities for combination studies that are discussed further below. 61 Lastly, autophagy may provide a temporary and finite source of arginine for cancer cells under amino acid restriction. Both ADI-PEG20 and arginase induce autophagy in thoracic cancer cell lines, which may be modulated using antimalarial agents such as chloroquine and quinine. 21,62 Notably, in a recent phase 1 study of ADI-PEG20-based combination therapy, a patient with relapsed glioblastoma multiforme exhibited prolonged re-sensitisation to arginine deprivation following the serendipitous prescription of quinine for leg cramps. 63,64 The clinical implications of autophagy modulators in the context of arginine deprivation for cancer therapy will require further study.

Strategic Combinations Incorporating Arginine Deprivation for SCLC Therapy
The disconnect between the marked preclinical efficacy of arginine deprivation in SCLC and an apparent lack of response of ADI-PEG20 monotherapy in patients may be explained by several factors, including tumor adaptation by ASS1 re-expression and autophagy, stromal refuelling and tumor heterogeneity. To overcome these hurdles, combination

Osteosarcoma Neuroblastoma
• ADI inhibits the proliferation of vascular endothelial cells • ADI inhibits proliferation by cell cycle arrest in G 1 and/or S phase leading to apoptosis 1999 Gong et al 106

Retinoblastoma Sarcoma
• ADI-PEG20 depletes plasma arginine (from 155μm to 2 μm) • ADI-PEG20 inhibits the production of NO treatments of ADI-PEG20 have now progressed into phase II and III studies in multiple cancers, and here we explore how these might be configured for SCLC as summarised in Figure 1.

Targeting DNA Repair
PARP are enzymes that play an important role in DNA repair by transferring ADP-ribose residues to targeted proteins. Some tumors depend highly on PARP for survival and therefore PARP inhibition is an interesting therapeutic strategy for these cancers. 55 PARP inhibitors (olaparib, veliparib, talazoparib) have been investigated for SCLC treatment. In 2019, Van Den Borg et al showed promising preliminary evidence of the preclinical and clinical activity of these molecules for SCLC treatment especially in sensitising cells to chemotherapy. 65 Moreover, in lymphoma, ADI-PEG20 treatment induced the activation of PARP cleavage, a marker of apoptosis, suggesting that arginine deprivation can have an impact on PARP derived pathways. 19 This highlights the potential for enhanced cytotoxicity by targeting the DNA damage/ repair pathway with arginine deprivation in SCLC.

Targeting DNA Synthesis and Replication
The high proliferative capacity of SCLC and the consequent demand for increased nucleotide precursors represents an area of potential combination studies with arginine deprivation. We showed that ADI-PEG20 downregulated thymidine salvage and de novo synthesis as a mechanism underlying potentiation with the anti-folate pemetrexed in ASS1deficient mesothelioma and bladder cell lines and consistent with the known diversion of aspartate for enhanced nucleotide synthesis in arginine auxotrophs. 31,66 Previous work in HCC cell lines revealed potentiation by arginase with the thymidylate synthase inhibitor 5-fluorouracil. 67 In addition to pemetrexed, ADI-PEG20 also synergised with the histone deacetylase inhibitor panobinostat in ASS1 deficient pancreatic ductal adenocarcinoma (PDAC). 68 The synthetic lethality was explained by the combination of accumulating DNA damage from panobinostat and cell cycle S-phase arrest upon ADI-PEG20 treatment in ASS1 deficient PDAC. In ASS1-ve cancer models, Prudner et al highlighted the efficacy of the combination of ADI-PEG20 with docetaxel and gemcitabine both in vitro and in vivo.
The study suggests that ADI-PEG20 with docetaxel translocate c-MYC to the nucleus thereby promoting hENT1 expression which induces gemcitabine uptake and increased cell death. 69 An ongoing phase II clinical trial is investigating the potential of this therapeutic combination for the treatment of sarcoma and SCLC (NCT03449901). Moreover, pemetrexed, gemcitabine and panobinostat have been studied in preclinical and clinical settings in SCLC raising the possibility of further combinations with arginine deprivation. [70][71][72][73] Similarly, arginine deprivation also modulates platinum and temozolomide cytotoxicity with evidence for additive and synergistic effects in arginine-auxotrophic cell lines. 17,74 While platinum forms the backbone of SCLC chemotherapy, temozolomide has documented activity in relapsed disease and further studies are warranted in combination with ADI-PEG20 and rhArgPEG. 75

Polyamines
Polyamines are highly regulated positively charged organic molecules that are found to be dysregulated in various cancers. Polyamines are essential for several cellular functions such as cell proliferation, apoptosis regulation, gene expression and protein translation. However, the mechanisms underlying these effects are yet to be fully described and understood. Locke et al showed the rewiring of the polyamine metabolism pathway in mesothelioma upon the appearance of ADI-PEG20 resistance. 24 Furthermore, combination treatment with a polyamine inhibitor reversed resistance to ADI-PEG20. Chalishazar et al highlighted the importance of the polyamine pathway in MYC-driven SCLC which was highly dependent on arginine regulation and sensitive to the polyamine biosynthesis inhibitor DFMO. 25 Efforts are ongoing to understand the role of the polyamine pathway in arginine auxotrophic cancers with the aim of developing novel combinations for testing in the clinic, including for patients with SCLC.

Glutamine Metabolism
Phosphoribosyl pyrophosphate amidotransferase (PPAT) transfers the gamma-nitrogen atom of glutamine to 5-phosphoribosyl pyrophosphate as the rate-limiting step of purine synthesis. RNA-sequencing of patients with SCLC revealed that PPAT expression was upregulated in cancerous cells compared to normal tissue whereas glutaminase was downregulated (GLS1). 76 These results indicate a rewiring of glutamine metabolism in SCLC and highlight the importance of this pathway. In melanoma and sarcoma, arginine deprivation induced by ADI-PEG20 caused a compensatory upregulation of the anaplerotic glutamine-glutamate axis refuelling the TCA cycle. This dependence was confirmed by the combination of ADI-PEG20 with GSL1 inhibitor BTPES that showed a synthetic lethal effect in these cell models. 22,27 Thus, the potency of a combination of ADI-PEG20 with a glutamine metabolism inhibitor is of interest in ASS1 deficient SCLC, thereby assessing the extent of glutamine metabolism rewiring upon arginine deprivation.

Hippo Pathway
Recent studies suggest four different molecular subtypes of SCLC depending on their neuroendocrine differentiation and gene expression profile. 5 SCLC-Y is driven by the expression of YAP1, a key regulator of the Hippo pathway. TEAD transcription factors mediate YAP1 activity to promote cell proliferation and growth. In SCLC, the YAP1 subtype is associated with a better prognosis compared to the other subtypes. 77 Several molecules, including verteporfin, decursin, amlexanox, and latrunculin, are known to inhibit YAP1-mediated transcription. Recent work in prostate cancer cells indicated that blocking TEAD activity potentiated the effect of arginine deprivation thus expanding these studies to SCLC are warranted. 78

PI3K/AKT/mTOR (PAM) Pathway
The PAM pathway is frequently dysregulated and active in SCLC in line with several other cancers. This pathway promotes cell growth and proliferation (via mTORC1) and cell cycle progression (via CDK4) while inhibiting apoptosis (via caspase-3). Various strategies are under development to target the recurrent alterations of this pathway

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in cancer, including inhibitors of the PAM pathway in combination with arginine deprivation, with clear applicability to SCLC. 79

Glycolysis and Warburg Effect
In SCLC, MYC-driven cancers display upregulated expression of glycolysis genes, correlating with a robust Warburg effect whereby the MYC-driven cells are less reliant on oxidative metabolism compared to cells with low MYC expression. 80 The use of glycolysis inhibitors such as PFK158 showed a significant decrease in glucose uptake, ATP production, and lactate for the MYC-driven cells, and controlled tumor growth in vivo. 80 ADI-PEG20 inhibited oxidative phosphorylation and aerobic glycolysis, therefore effectively inhibiting the Warburg effect in melanoma and sarcoma cell lines. Hence, further testing of ADI-PEG20 with inhibitors of glycolysis may provide a novel metabolic combination strategy for MYC-driven SCLC.

Immunotherapy Combinations
SCLC is a highly mutated cancer with a history of heavy tobacco use in 98% of patients, the highest for any malignancy. Consequently, SCLC is immunologically tractable with recent studies defining a role for programmed death-ligand 1 (PD-L1) antagonists in combination with chemotherapy. 1 However, despite the high tumor mutation burden, the benefits of immune checkpoint therapy have been modest, implying a role for extensive tumor-induced immunosuppression. Recent single cell transcriptome data have revealed marked T cell dysfunction -a low ratio of CD8+ effector to Treg cells -and enrichment of profibrotic macrophages linked to exhausted CD8+ T cells as major drivers of the immune cold TME. 81 An anticancer immune response is dependent on multiple factors, with arginine playing pivotal roles especially in maintaining T cell receptor function, proliferation and memory. 82 Critically, nitric oxide synthase (NOS) via the generation of NO, and arginase (1 and 2) via the production of ornithine, are major arginine catabolizing enzymes involved in orchestrating cellular crosstalk and thereby immune and inflammatory responses. 83,84 Recently, low plasma levels of arginine have been linked to poor outcomes in patients receiving immune checkpoint inhibitors, whereas high circulating arginine was linked to improved disease control, further emphasizing the potential of arginase inhibitors as cancer therapeutics. 85 Nonetheless, preclinically, there is also a good rationale for combining immune checkpoint immunotherapy with arginine deprivation as shown in Figure 2. ADI-PEG20 increases tumoral PD-L1 expression and T cell infiltration and reduces Treg cell accumulation with potentiation of PD-1/PD-L1 inhibitors in arginine auxotrophic syngeneic tumor murine models. 86 Similarly, Co-Arg1-PEG has shown enhanced tumor control in vivo in combination with PD-1/PD-L1 inhibitors and anti-OX40 antibodies. 87 Moreover, analysis of urea cycle dysfunctional cancer predicts for gene signatures linked to immune responsiveness. 88 Additionally, targeted antigens for chimeric antigen receptor (CAR) T-cell-based therapy in preclinical and clinical studies of SCLC, namely DLL-3, CD55 and GPC3, may be enhanced through metabolic engineering of urea cycle enzymes, ASS1 and OTC, thereby maintaining T cell immunocompetency. 89 Additionally, citrulline the byproduct of ADI-PEG20, may be recycled by T cells to regenerate arginine thereby maintaining immune cell function, a critical consideration with immune checkpoint inhibitors. 90 Clinically, ADI-PEG20 was tested with pembrolizumab in a small phase 1b study of patients with advanced treatment-refractory cancers, mostly aerodigestive in origin, and none with lung cancer. 91 The combination was safe and active, documenting a partial response rate of 24% and a grade 3-4 neutropenia rate of 40%, which is notable in that anti-PD-1 inhibitors rarely cause neutropenia and this occurs infrequently with ADI-PEG20 monotherapy. 29,41,42,59 Repeat biopsies on ADI-PEG20 -and prior to commencement of pembrolizumab -revealed infiltration by CD3+ T cells consistent with the pre-clinical data. Results are also expected from a phase I/II study of pegzilarginase combined with pembrolizumab which included a SCLC dose-expansion cohort (ClinicalTrials.gov Identifier: NCT03371979).

Current Therapy for Small Cell Lung Cancer and Outcomes
Whilst in general SCLC responds robustly to initial platinum-based chemotherapy, the majority of patients subsequently relapse. 92,93 The chance of responding to second-line treatment is approximately 10% and resistance emerges rapidly in https://doi.org/10.2147/LCTT.S335117

DovePress
Lung Cancer: Targets and Therapy 2022:13 nearly all patients. 94 Clinically, the terms "limited disease", and "extensive disease" are used to define the extent of SCLC burden. Limited disease is defined as the tumor being confined to one hemithorax and regional lymph nodes and treatment options include surgical resection in a minority aiming for R0 resection, concurrent chemotherapy (with platinum-based chemotherapy in combination with the topoisomerase II inhibitor, etoposide) and thoracic radiotherapy. Prophylactic cranial irradiation (PCI) serves to decrease the risk of symptomatic brain metastases and increases overall survival in patients who are Performance status 0-1 and experience a complete remission. 95 Despite this, the median OS of limited-stage SCLC is approximately 17 months.
Patients with "extensive disease" are treated with platinum-based chemotherapy usually combined with etoposide with a resultant median overall survival (OS) of 9-10 months, progression free survival (PFS) of 5-6 months and 1 year OS of ~35%. 96 A modest advance in the last 40 years in the first-line treatment setting of SCLC has been the advent of chemoimmunotherapy with the addition of an anti-programmed death-ligand 1 (PD-L1) antibody based on the IMpower133 and CASPIAN studies. [97][98][99] The IMpower 133 trial tested the addition of the anti-PD-L1 antibody, atezolizumab to carboplatin and etoposide for 4 cycles followed by atezolizumab maintenance versus placebo and carboplatin and etoposide. Patients in the atezolizumab arm achieved a median OS of 12.3 months compared to 10.3 months in the placebo arm (HR = 0.70; 95% CI 0.54 to 0.91 p = 0.007) and with almost 2 years of median follow-up, patients in the atezolizumab arm continued to display benefit with ~22% of patients alive at 24 months in the atezolizumab arm, compared with ~14% in the placebo arm. 97 The CASPIAN trial enrolled 805 patients to durvalumab with or without the anti-cytotoxic T-cell lymphocyte-4 blocking antibody, tremelimumab, plus platinum-etoposide followed by durvalumab maintenance or platinum-etoposide only. The combination of platinum-etoposide plus durvalumab significantly extended OS, compared with platinumetoposide alone (HR = 0.73) 95% CI, CI 0.59 to 0.91; p = 0.0047. Furthermore, patients in the durvalumab/platinum/ etoposide arm had a sustained OS benefit; an additional 8% of patients were alive at 24 months in the experimental arm (22.2%) compared with the platinum/etoposide arm (14.4%). The addition of tremelimumab provided no additional survival benefit. 98 While KEYNOTE-604, which tested the role of pembrolizumab with platinum-etoposide, significantly improved PFS, the pre-specified endpoint for OS was not met. 100 Despite these recent therapeutic developments with the addition of anti-PD-L1-based immunotherapy to standard chemotherapy for SCLC, the prognosis remains poor and new strategies are clearly required to go beyond the current therapeutic plateau.
Thus, in view of the modest gains obtained by incorporating anti-PD-L1 blockade to the carboplatin-etoposide chemotherapy backbone, there is clear scope for further improving survival outcomes for patients with SCLC. Arginine deprivation is a particularly attractive anti-metabolite option that on current evidence is safe in triplet chemotherapy combinations and as a doublet with immune checkpoint blockade. Indeed, in view of the preclinical data described above, there are potential synergies to explore for both the chemotherapy and immunotherapy components as a quadruplet chemo-immunometabolic therapy with arginine depletion.

Perspectives for Arginine Deprivation in SCLC
ATOMIC-meso is the first front-line triplet chemotherapy phase III study of arginine deprivation using ADI-PEG20 combined with platinum and pemetrexed in cancer, specifically non-epithelioid mesothelioma, and is expected to report by the summer of 2022. Based on the premise that ADI-PEG20 sensitises ASS1-deficient cancers to antifolates by exploiting a critical dependency on nucleotide synthesis, ATOMIC-meso, if positive, will herald the advent of arginine deprivation as a novel antimetabolite therapy for cancer.
As alluded to earlier, there is significant potential to leverage arginine auxotrophic SCLC with arginine deprivation in combination with chemoimmunotherapy, namely platinum plus etoposide and an anti-PD-L1 agent which would be a rational pathway to development. Other combinations may have merit particularly replacing etoposide for pemetrexed in patients with reduced performance status. Efforts are also underway with a phase II trial investigating ADI-PEG20 in combination with gemcitabine and docetaxel, that includes a small cohort of patients with SCLC. (NCT03449901). Additional agents that warrant investigation in relapsed SCLC based on a study in progress in glioblastoma multiforme, include ADI-PEG20 with temozolomide with or without radiation (NCT04587830).

Conclusions
There are significant opportunities ahead to develop our understanding of the role of arginine deprivation as part of combined multimodality care for patients with SCLC. More broadly, arginine depletion as an antimetabolite strategy should be reinterpreted both in cancer treatment and in reshaping the tumor microenvironment, with a specific focus on tumor-type. Ultimately, a strong translational base will be needed to decipher which SCLC subgroups benefit the most from incorporating arginine depletion to the standard of care.

Disclosure
Dr Szlosarek reports grant research support from Polaris Group. The remaining authors report no other conflicts of interest.


Isoschaftoside Reverses Nonalcoholic Fatty Liver Disease via Activating Autophagy In Vivo and In Vitro

Nonalcoholic fatty liver disease (NAFLD) is the most common metabolic liver disease globally, and the incidence of NAFLD has been increasing rapidly year by year. Currently, there is no effective pharmacotherapy for NAFLD. Therefore, studies are urgently needed to explore therapeutic drugs for NAFLD. In this study, we show that isoschaftoside (ISO) dramatically reduces lipid deposition in cells. Meanwhile, ISO treatment reverses the NAFLD and reduces hepatic steatosis in mice. Importantly, we reveal that ISO suppresses the expression of light-chain 3-II (LC3-II) and SQSTM1/p62 in palmitic acid (PA) induced autophagy inhibition in the cell model and the NAFLD mouse model, which suggests that ISO might reverse NAFLD through regulating autophagy flux. We propose that ISO might alleviate hepatic steatosis in NAFLD via regulating autophagy machinery. Consequently, our study suggests that ISO might be of potential clinical value in the field of NAFLD therapy. ISO might have the potential for future therapeutic application.

Introduction
NAFLD is a disease characterized by liver parenchymal cell steatosis and intracellular lipid storage [1], which is usually connected with insulin resistance, obesity, diabetes, and other diseases [2]. NAFLD also affects the metabolism of substances in the body, which can bring about a range of changes in blood lipid and blood sugar levels [3,4]. e morbidity of NAFLD has been rising year by year in both developed and developing countries [5]. e prevalence of NAFLD is increasing in all populations. More seriously, NAFLD can develop into chronic hepatitis, cirrhosis, and finally liver cancer [1]. Although lots of studies have been done on NAFLD, the pathogenesis of NAFLD is too complex to be fully elucidated. To date, there is still a lack of accurate and efficient curative drugs for NAFLD treatment in clinics. erefore, finding an effective treatment for NAFLD is a good choice.
It was proposed that lipid metabolism disorders lead to lipid accumulation in liver cells [6]. Additionally, autophagy is a crucial mechanism to regulate lipid metabolism [7]. e autophagy-regulating lipid metabolism in the liver is called lipophagy, and lipids can be decomposed into free fatty acids through lipophagy, which can be reused by the body [8]. It has been reported that the autophagy in NAFLD could be inhibited in the presence of a high-fat diet or inducers, which results in lipid dysfunction and impaired autophagy flux, leading to lipid deposition in the liver [9].
Recently, many studies have demonstrated that the extracts of Chinese herbal medicine could activate autophagy to improve the severity of fatty liver and reduce steatosis and liver inflammation [10,11]. e application of Chinese herbal medicine promises to be an important means of treating NAFLD. However, its specific mechanism of action in the field still needs further investigation. Studies indicated that the therapeutic treatment of NAFLD by Chinese herbal medicine has attracted more and more attention [12]. Chinese herbal medicine is a kind of natural ingredient extracted from plants. Compared with Western medicine, it has relatively lower rates of toxicity and side effects. Studies have shown that effective ingredients extracted from Chinese herbal medicine have been demonstrated to play important roles in the clinical effectiveness of NAFLD treatment. Clinical studies proposed that Chinese herbal medicine achieved a good clinical therapeutic effect in NAFLD treatment [13]. To date, the Chinese herbal extracts were shown to relieve NAFLD in numerous aspects, such as lipid metabolism abnormalities, endoplasmic reticulum stress, oxidative stress, inflammation, hepatocyte apoptosis, and necrosis [3,4]. Silybin and berberine have been used in phase 4 clinical studies in the United States [14]. Silybin can reverse NAFLD by reducing oxidative stress and inflammatory response [15]. Berberine has a certain effect on endoplasmic reticulum stress and inflammation in NAFLD [16]. Recent studies have shown that extracts of Chinese herbal medicine can induce autophagy regulation, which is one of the relevant mechanisms involved in NAFLD. erefore, Chinese herbal medicine-induced autophagy activation is a direction for NAFLD therapeutic treatment.
Isoschaftoside (ISO, C26H28O14; Supplementary  Figure 1(a)) is a Chinese herbal monomer extracted from Abrus cantoniensis Hance, one of the flavonoids' active components [17]. Flavonoids are a kind of Chinese herbal medicine for the treatment of NAFLD. e active components of total flavonoid glycoside C include vicenin-2, isoschaftoside, and schaftoside [18]. Studies have shown that vicenin-2 could regulate inflammatory pathways in the treatment of NAFLD [19]. e studies published in the Chinese Journal of Traditional Chinese Medicine (2017, 32 (11):5078-5081) proposed the protective effect of schaftoside on NAFLD induced by a high-fat diet in mice. e vicenin-2 and schaftoside have been shown to have therapeutic effects on NAFLD. erefore, we speculated that isoschaftoside might have therapeutic effects on NAFLD. Studies have shown that in traditional Chinese medicine Abrus cantoniensis Hance has the function of soothing the liver and promoting gallbladder and can be used to treat hepatitis [20,21], but the specific effective composition is not clear. To date, the research on the effects of ISO is still at an early stage. In this study, we demonstrated that ISO could reverse hepatic steatosis in NAFLD through regulating autophagy flux.

Cell Culture and Treatment.
HepG2 cells were purchased from Procell Life Science & Technology Co., Ltd. e cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin (Gibco, USA) in a 37°C 5% CO 2 incubator. e preparation of 10 mM PA is referred to the existing literature [22]. Sodium palmitate in PBS was incubated at 70°C for 30 minutes and mixed with 30% BSA in PBS at a 1 : 1 volume ratio of PA/BSA. 0.4 mM PA was used to induce autophagy cell models. 15% BSA was used as a blank control. We labeled blank control as the NC group. To establish the cell model of NAFLD in vitro, PA was added into the medium at a concentration of 0.4 mM. In in vitro experiment, we used different concentrations of ISO and 0.4 mM PA or equivalent BSA solution to culture cells for 24 h.

CCK-8 Assay.
We seeded 5 × 10 3 HepG2 cells on 96-well plates. After 24 h, the medium was replaced with fresh medium with various concentrations of PA. Following 24 h of treatment, we added 10 μl CCK-8 solution and 100 μl fresh medium into each well. e absorbance was measured by spectrophotometer at 450 nm wavelength after 2 h of treatment.

Oil Red O Staining to Evaluate the Level of Lipid
Deposition. Oil Red O Staining of Cells: after washing the cells twice with PBS, we fixed the cells with 4% paraformaldehyde for 10-15 minutes and stained the cells with the Oil Red O Kit; Oil Red O Staining of Liver Tissues: liver tissues were immediately frozen in liquid nitrogen after harvesting. en, the samples were embedded. e frozen liver slices were prepared and stained with Oil Red O Staining Kit. ree random images were captured using a light microscope (Olympus BX53) for each sample.

Western Blot.
e total protein was extracted with RIPA buffer (containing 0.1% SDS, 1% protease inhibitor, and 1% phosphatase inhibitor; Solarbio, R0020). Protein quantification was performed using the BCA Kit (Coolaber, SK1070-5000T). 20 μg protein lysis was prepared for SDS-PAGE (6% concentrated gel and 12% and 15% separated gel) and transferred to 0.22 μm Immobilon-PSQ PVDF membrane (Merck Millipore Ltd., ISEQ00010). e membranes were blocked with 5% nonfat-dried milk (Coolaber, CN7861-500G) at room temperature for 2 h, washed 3 times with TBST (Solarbio, No. T1080) for 15 minutes each time, and hatched with primary antibody at 4°C overnight. After being washed, the membranes were hatched with the corresponding secondary antibody at room temperature for 1 h and then washed 3 times with TBST for 15 minutes each time. β-actin was used as an internal control. All immunoreactive protein bands were visualized by BeyoECL Moon (Beyotime, P0018FM-2) and quantified by software Image-Pro Plus 6.0.

Animal Experiments.
We conducted NAFLD animal model using C57 mice fed with a high-fat diet [23,24]. 20 C57 mice (4 weeks; male; C57BL/6JGpt) were purchased from Jiangsu Jicuiyaokang Biotechnology Co, Ltd., and all mice were kept in a pathogen-free facility at a specific temperature (24°C ± 5°C), a specific humidity (55% ± 5%), and a specific light (12 h of light/12 h of dark cycle). After 1 week of adaptive feeding, they were randomly divided into two groups. e control group (n � 6) was fed with an ordinary diet, and the high-fat diet group (n � 14) was fed with a high-fat diet, and their weight was recorded weekly for 16 weeks. Starting from the 17th week, the high-fat diet group was randomly divided into the HFD group (n � 7) and the ISO-treated group (n � 7). We considered the HFD group as the model group and continued to feed the HFD group and the ISO-treated group with a high-fat diet for 4 weeks. e ISO-treated group was given ISO (20 mg/kg/day) by intraperitoneal injection, and the control group and the HFD group were given equal 0.9% normal saline for 4 weeks. e body weight of mice was recorded weekly. All animal experiments were carried out in accordance with the Experimental Animal Welfare Act and approved by the Experimental Animal Welfare Ethics Committee. Our laboratory has a laboratory animal license.

Blood Biochemical
Test. Blood samples were collected from each mouse by eyeball sampling. e serum was centrifuged at 3000 rpm for 10 minutes at 4°C and stored at −80°C.

Hematoxylin and Eosin (H&E) Staining. All fresh livers
were washed with normal saline and fixed in a 10% neutral paraformaldehyde buffer solution. Paraffin-embedded liver biopsy sections (5 mm) were stained with H&E for histological analysis. e sections were observed and taken pictures with a light microscope (Olympus BX53) for changes in the organization structure.

Statistical
Analysis. All data were represented by the mean ± standard deviation (SD) in three independent trials. e Mann-Whitney U test (nonnormally distributed variables) and t test (normally distributed variables) were used for comparison between two groups; the Kruskal-Wallis H test (nonnormally distributed variables) and one-way analysis of variance were used for comparison between multiple groups. Data were analyzed by GraphPad Prism 7.0 software. All data were represented by the mean ± standard deviation (SD) in three independent trials. * P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001.

ISO Reduces Lipid Deposition in Cells via Activating
Autophagy Flux. To investigate the therapeutic effect of ISO in NAFLD, we employed the cell model to evaluate the regulatory capacity of ISO in lipid deposition. e cytotoxicity assessments of ISO and PA were performed using a CCK-8 assay.
e HepG2 cells were treated with a concentration gradient of ISO or PA. We observed that ISO had no significant impact on cell proliferation at a concentration below 500 μM (Figure 1(a)). We used the Caspase-3 Activity Assay Kit to examine the variation of apoptosis of cells after ISO treatment. We found that ISO treatment did not affect cells significantly (Supplementary Figure 2(b)). As reported previously, 0.4 mM PA was used to repress autophagy in HepG2 cells [25,26]. We also found that PA exhibited a dose-dependent induction of cytotoxicity in HepG2 cells ( Figure 1(b) i), IC50 � 0.5861 ± 0.00282 mM (Figure 1 To examine the PA-induced inhibition of autophagy flux, we examined p62, LC3-II, beclin-1, and ATG5 protein expression level using Western blot. LC3-II and p62 were the proteins related to autophagy substrates, and the increased expression of them indicated that the function of autophagy was inhibited. e expression levels of p62 and LC3-II increased as the concentration of PA increased, while no significant changes in the expression levels of beclin-1 and ATG5 were observed after PA treatment, and we used β-actin as an internal reference for quantitative analysis of autophagy-related protein (Figure 1(c)). Quantification of Western blot was performed using ImageJ (Supplementary Figure 1(b)). erefore, we speculated that PA might block the fusion process of autophagosome and lysosome, leading to the accumulation of autophagic substrate proteins LC3-II and p62, while PA had no significant effect on the formation of autophagosome [27]. We suggested that PA might inhibit the downstream pathway of autophagy resulting in lipid deposition. e results revealed that we successfully established the PA-induced autophagy inhibition model in HepG2 cells in our study. Meanwhile, 0.4 mM of PA treatment was determined to be used for PA-induced autophagy inhibition model, which significantly inhibited autophagy with a relative lower cytotoxicity in HepG2 cells. We then treated cells with a concentration gradient of ISO Evidence-Based Complementary and Alternative Medicine and examined the effect of ISO treatment on autophagy in HepG2 cells. Autophagy flux was detected by Western blotting using LC3-II and p62 antibodies (Figure 1(d)). When the concentration of ISO treatment was higher than 200 μM, we observed significant reductions in LC3-II expression. e results suggested that the autophagy was activated when the concentration of ISO treatment was increased to 200 μM. We then examined the effect of ISO treatment on PA-induced autophagy inhibition in HepG2 cells. As shown in Figure 1

Experimental Evidence for Autophagy Activation by ISO.
To further investigate the mechanisms of ISO-reduced lipid deposition via activating autophagy, we measured the expression of autophagy-related proteins such as LC3-II, p62, beclin-1, ATG5, mTOR, and p-mTOR in cells after different treatments. We observed that the expressions of LC3-II and p62 increased after PA treatment, and the increased expression of LC3-II was synchronized with the accumulation of p62, indicating that the autophagy flux was impaired (Figure 2    Under the same experimental conditions, we further verified the association between autophagy and lipid deposition by inhibiting autophagy through CQ [28]. CQ is an inhibitor of autophagy, which can block the fusion of autophagosome and lysosome, thereby inhibiting the autophagy flux [28,29]. We cultured HepG2 cells separately with CQ and then performed Oil Red O staining and Western blot to examine lipid deposition and autophagy flux. In the Oil Red O staining, compared with the control group, lipid deposition was significantly increased in the CQ group, indicating that CQ inhibited the autophagy flux of HepG2 cells, leading to lipid deposition (Figure 2(b), control group vs CQ group). In general, the inhibition of autophagy flux can lead to lipid deposition [30,31], and the activation of autophagy flux can reduce lipid deposition, which also provides a theoretical basis for our experiment.

ISO Treatment Decreased Liver Steatosis in High-Fat Diet
Mice. To investigate the effects of ISO on high-fat diet mice, C57BL/6 mice were fed a standard purified rodent diet or a high-fat diet, respectively, for 16 weeks and then given ISO (20 mg/kg/day) or equivalent normal saline for 4 weeks (Figure 3(a)). From the first week to the 16th week, we recorded the changes in the body weight of the mice and conducted the statistical analysis. e experimental results showed that the body weight of mice in the HFD group was significantly higher than that in the control group, suggesting that the high-fat diet caused obesity in mice compared with the control group (Figure 3(b)). Based on the weight changes in the control group and the high-fat diet group, we believed that the establishment of the NAFLD animal model was successful. To explore the effect of ISO intervention on high-fat diet mice, we randomly divided the high-fat diet mice into two groups, and we got the HFD group and the ISO-treated group. e HFD group was the vs. ISO-treated group). is result indicated that ISO could reduce the body weight of high-fat diet mice, which was a vital basis for ISO treatment of NAFLD. After anesthesia, the mice were weighed and photographed. From the morphology of the mice, the HFD group was fatter than in the control group (Figure 4(a) i, control group vs. HFD group), and the results of body weight showed that the HFD group was significantly heavier than in the control group (Figure 4(a) ii, control group vs. HFD group). Particularly, ISO significantly reduced the body weight and body size of the high-fat diet mice compared with the model group (Figure 4(a) i, ii, HFD group vs. ISO-treated group). Meanwhile, we know that hepatic steatosis is closely associated with increased visceral fat [32]. After dissecting the mice, the visceral fat weight of the HFD group was significantly higher than that of the control group (Figure 4(a) iii, control group vs. HFD group), but ISO could significantly reduce the visceral fat weight of high-fat diet mice (Figure 4(a) iii, HFD group vs. ISO-treated group). According to the morphology of mice liver, the color of the livers in the HFD group was whiter than that in the control group (Figure 4(b) i, control group vs. HFD group), and the morphological changes in livers suggest that the high-fat diet caused liver steatosis in mice. e livers of the mice on the high-fat diet were significantly larger than those of the control group, suggesting that the high-fat diet not only increases body weight but also increases liver size. So, the body weight, liver weight, and visceral fat weight of mice in the high-fat diet group were significantly higher than those in the control group. On these indicators, liver steatosis was more severe in the high-fat diet group than in the control group. ese indicators were reversed after the ISO-treated group compared with the HFD group (Figure 4(a) and 4(b), HFD group vs. ISO-treated group), suggesting that ISO treatment could reverse liver steatosis induced by a high-fat diet (Figure 4(b), HFD group vs. control group, control group vs ISO-treated group). We speculated that ISO treatment might reduce the liver index. In H&E staining, we observed that there were obvious ballooned lipid vacuoles in the liver tissues of the high-fat diet group (Figure 4(c), control group vs HFD group), indicating that there were lipid deposits in the liver tissues. e lipid deposits were significantly mitigated after ISO treatment (Figure 4(c), HFD group vs. ISO-treated group). is phenomenon was further confirmed by the Oil Red O staining and frozen section (Figure 4(c), control group vs. HFD group, HFD group vs. ISO-treated group). ese liver histological changes demonstrated that ISO could significantly attenuate hepatic lipid accumulation induced by a high-fat diet and decrease the number of intracellular lipid droplets and hepatocyte balloons. ese results suggested that ISO could improve body weight, liver weight, and fat accumulation in high-fat diet mice, and we suggested that ISO had a dramatic therapeutic effect on NAFLD.

ISO Improves Liver Function and Blood Glucose in High-
Fat Diet Mice. Studies have shown that liver steatosis will be accompanied by changes in blood indicators [33]. To explore the effects of ISO on the function of the liver and blood glucose in mice, we extracted serum from mouse blood and tested them with various blood index kits. We selected and examined ALT, AST, GLU, TC, and TG as a reference and tested the serum of mice ( Figure 5(a)). e results of blood biochemical analysis showed that the levels of these indicators in the HFD group were significantly higher than those in the control group ( Figure 5(a) i, ii, iii, iv, v, control group vs. HFD group), indicating that the high-fat diet led to liver function injury and raised blood glucose, but the blood biochemical indicators in the ISO-treated group were lower than those in the HFD group ( Figure 5(a) i, ii, iii, iv, v, HFD group vs ISO-treated group).
ese experimental results suggested that ISO reversed the damage to liver function and the elevation of blood glucose caused by a high-fat diet. erefore, we believed that ISO could improve liver function damage and insulin resistance caused by a high-fat diet, indicating that ISO had therapeutic potential on NAFLD.

ISO Attenuates the Inhibitory Effect of a High-Fat Diet on
Autophagy.
rough the above results, we observed that ISO improved liver steatosis caused by a high-fat diet, and we further learned that autophagy played a significant role in the treatment of NAFLD [9]. Next, we explored whether ISO could reduce liver steatosis in mice on a high-fat diet by activating autophagy. We extracted proteins from mice liver tissue and observed the changes in autophagy-related proteins. We examined the expression levels of the proteins using Western blot ( Figure 5(b)), and the expression of p62 and LC3-II in the HFD group was significantly higher than in the control group ( Figure 5(b), control group vs. HFD group). e expression level of p62 and LC3-II in the ISOtreated group was significantly lower than that in the HFD group ( Figure 5(b), HFD group vs. ISO-treated group). ISO reduced the expression of p62 and LC3-II, suggesting that ISO improved autophagy inhibited by the high-fat diet in vivo. ISO might promote the degradation of autophagylysosome and thus reduce the accumulation of p62 and LC3-II protein. However, there was no significant difference in beclin-1 and ATG5 among the three groups ( Figure 5(b), HFD group vs. control group, HFD group vs. ISO-treated group, control group vs. ISO-treated group), indicating that a high-fat diet and ISO treatment did not affect autophagosome formation.

Discussion
Recently, more and more people have paid attention to the treatment of NAFLD by Chinese herbal medicine [13]. Many drugs for the treatment of NAFLD were also extracted from Chinese herbal medicine, such as silymarin and berberine, which had been widely used to treat NAFLD [34,35]. Many clinical studies also supported NAFLD treatment by Chinese herbal medicine. A growing number of studies supported the treatment of NAFLD in Chinese herbal medicine [36,37].
e Chinese herbal medicine of flavonoids had anti-inflammatory and antioxidant stress effects, which provided a theoretical basis for treating NAFLD by flavonoids [17,38]. 8 Evidence-Based Complementary and Alternative Medicine ISO is a monomer of traditional Chinese medicine extracted from Acacia chinensis and is one of the active components of total flavonoid glycosides. ere were few studies on the treatment of NAFLD by ISO. e main content of our study was that ISO reversed NAFLD by activating autophagy flux in vivo and in vitro.
At present, the pathogenesis of NAFLD is still largely unknown, and our in vivo and in vitro study provides an excellent theoretical basis. We found that ISO had a strong liver protective effect on NAFLD, and we considered that autophagy might be involved in the effect. Recently, we have seen that autophagy played an irreplaceable role in hepatic steatosis [39]. We conducted a series of experiments to verify this phenomenon.
We conducted a study with HepG2 cells. We found that PA-induced lipotoxicity played an essential role in the pathogenesis of NAFLD [25,40]. We treated HepG2 cells with PA to establish the NAFLD lipid deposition cell model. It has been suggested that HepG2 cells can be used as a cell model for lipid deposition [27]. HepG2 cells treated with different concentrations of PA resulted in the accumulation of p62 and increase in LC3-II, but beclin-1 and ATG5 related to autophagosome formation did not change significantly, indicating that cell lipid deposition induced by PA did not affect autophagosome formation but inhibited the degradation of autophagy lysosomes, resulting in downstream damage of autophagy pathway. In short, ISO could reduce the accumulation of LC3-II and p62 after PA treatment, but ISO did not affect the expression of beclin-1 and ATG5. We further verified the role of ISO in reducing hepatic steatosis and activating autophagy flux in vivo. We found that in mice ISO treatment significantly reduced hepatic steatosis, TG, TC, AST, ALT, and GLU levels. Compared with the HFD group, the liver lipid deposition was significantly reduced in the ISO-treated group, and Oil Red O staining showed a marked decrease in intracellular lipid droplets in liver tissue. ese experiments suggest that ISO treatment could reverse the degree of hepatic adipose degeneration. Compared with the HFD group, the body weight, liver weight, liver index, and visceral fat weight of C57 mice in the ISO-treated group were decreased. ese results suggest that ISO has a protective effect on NAFLD.
e autophagy of the liver in C57 mice fed with a highfat diet was impaired [27]. e accumulation of p62 and the increase in LC3-II protein occurred simultaneously in the liver of C57 mice, indicating that the function of autophagy in C57 mice liver was impaired [41]. ISO treatment reduced the expression of p62 and LC3-II, which suggested that ISO could improve the inhibition of autophagy in the liver of mice. rough literature reviews, we found that the accumulation of p62 reflected the decrease in autophagy flux. Conversely, the activation of autophagy flux could decrease p62 expression [42]. LC3-II was also an autophagy substrate protein, and the increased expression of LC3-II could not simply be considered as autophagy activation or inhibition. It needed to evaluate the autophagy state together with p62 [43]. e accumulation of LC3-II might be interpreted as a result of inducing autophagy or blocking downstream of autophagy [43]. erefore, in our study we considered that the decreased expression of p62 and LC3-II in ISO treatment might reflect the activation of autophagy flux.
According to literature studies, the mechanisms of increasing autophagy flux are as follows: firstly, promoting autophagosome formation; secondly, increasing the fusion of autophagosome and lysosome into autophagolysosome [43]. We found that ISO treatment reduced LC3-II and p62 levels but had no effect on the expression of beclin-1 and ATG5. erefore, we believe that ISO is unlikely to activate autophagy by promoting upstream autophagosome formation. ISO may improve autophagy flux by increasing autophagosome-lysosome fusion, and ISO may play a role downstream of autophagy. e activation of autophagy was helpful for the treatment of NAFLD. Autophagy activation could rapidly metabolize the lipid accumulation in liver cells, and autophagy played a vital role in liver lipid metabolism [44]. e imbalance of autophagy was closely related to the occurrence and development of liver diseases, and people had found that autophagy could also affect liver cirrhosis and even liver cancer [45]. Recent studies have shown that autophagy is closely associated with obesity and insulin resistance [46], which may also be a momentous factor in accelerating the progression of NAFLD after autophagy is affected.
In this experiment, it may be necessary to further observe the number of intracellular lipid droplets and autophagosomes in cells or liver tissues by scanning electron microscopy, to further explore the reversal of NAFLD through activation of autophagy by ISO. Finally, we summed it up. e results of in vivo and in vitro experiments showed that ISO might reverse hepatic steatosis through regulating the fusion of autophagosome and lysosome. e activation of autophagy can be used as a treatment for hepatic steatosis.
ese results suggest that ISO-mediated autophagy regulation may provide a novel strategy for the treatment of NAFLD.

Conclusion
In conclusion, our study showed that ISO reduced PA-induced intracellular lipid droplets in HepG2 cells in vitro, and ISO reversed liver steatosis induced by a high-fat diet in vivo, which might be related to ISO activation of the autophagy pathway. Our study suggested that ISO might be a potential treatment for NAFLD.

Data Availability
e data used to support the results in this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.


s2fieldsofstudy: Biology Chemistry

text:
Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveal a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary, we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.

The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Molecule 1  3 Residue-property plots i O These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.
• The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 4.
All (31)  There are no Ramachandran outliers to report.

Protein sidechains i O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are dened in the Chemical Component Dictionary. The Link column lists molecule types, if any, to which the group is linked. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, 7O36 bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included. For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier. Any bond that is central to one or more torsion angles identied as an outlier by Mogul will be highlighted in the graph. For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identied by Mogul is calculated over all ring torsion angles. If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identied rings is also greater than 60 degrees, then that ring is considered an outlier. The outliers are highlighted in purple. The color gray indicates Mogul did not nd sucient equivalents in the CSD to analyse the geometry.
Ligand GTP D 401 The following is a graphical depiction of the model t to experimental electron density of all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included. Each t is shown from dierent orientation to approximate a three-dimensional view.
Electron density around GTP A 401:


s2fieldsofstudy: Biology Medicine

text:
Electroacupuncture Reduces Cerebral Hemorrhage Injury in Rats by Improving Cerebral Iron Metabolism

Objective To study the effects of electroacupuncture at Baihui and Dazhui points on the expression of hepcidin (Hepc), transferrin (Tf), transferrin receptor (TfR), and ferritin (Ft) in rats with cerebral hemorrhage to provide a theoretical basis for the treatment of cerebral hemorrhage with acupuncture. Method The model of cerebral hemorrhage in rats was established by autologous blood injection method and treated by electroacupuncture (EA) at the acupoints of Baihui and Dazhui. Hepc siRNA was injected into the lateral ventricle 30 min before model preparation to produce the cerebral hemorrhage model. The modified neurological severity score (mNSS) was used to assess the neurological function, and the total iron content in brain tissue was determined using atomic absorption spectrometry; the expression of Hepc, Ft, Tf, and TfR in perihematoma tissue was detected using immunohistochemistry; the interference efficiency of Hepc siRNA was detected using western blot and reverse transcription polymerase chain reaction (RT-PCR). Results The degree of neurological deficit showed a downward trend at 3 days, 7 days, and 14 days, and electroacupuncture significantly reduced the neurological deficit score at each time point (P < 0.01). Regarding total iron content in brain tissue, on the 3rd day, the 7th day, and the 14th day, the iron content of the hematoma tissue after intracerebral hemorrhage was reduced by electroacupuncture (P < 0.01). Regarding immunohistochemical results. Hepc, Ft, Tf, and TfR protein expressions on day 14 were significantly higher after cerebral hemorrhage (P < 0.01). After electroacupuncture, the expression of Hepc, Ft, Tf, and TfR protein was significantly reduced (P < 0.01). Western blot and RT-PCR revealed that the interference efficiency of Hepc siRNA was statistically significant (P < 0.01). Conclusion Electroacupuncture can reduce neurological severity scores in rats with cerebral hemorrhage and may exert cerebral protective effects by reducing Hepc protein and gene expression; lowering Ft, Tf, and TfR protein expression; and promoting iron metabolism in the brain of rats with cerebral hemorrhage.

Introduction
Cerebral hemorrhage (ICH) is a subtype of stroke that accounts for approximately 10-20% of all strokes and has a 1-month mortality rate of 20-40%, with higher incidence, morbidity, and mortality than those of ischemic stroke [1][2][3]. The important factors contributing to the poor prognosis of patients are secondary injuries after cerebral hemorrhage, including apoptosis, inflammatory response, tissue necrosis, erythrocyte lysis, and cerebral edema [4,5]. In recent years, it has been found that iron deposited in the tissue surrounding the hematoma after cerebral hemorrhage is an important factor contributing to secondary brain injury [6]. After cerebral hemorrhage, hemoglobin is broken down into iron ions and bilirubin by heme oxygenase 1 (HO-1), the rate-limiting enzyme of heme catabolism, which has strong oxidative properties and plays an important role in pathological responses such as neuronal cell injury and secondary brain edema [7,8]. Among them, hepcidin (Hepc) is an essential iron-regulating peptide hormone that plays an important role in maintaining the balance of iron metabolism in the body and cells by regulating intestinal iron absorption, serum iron concentration, and tissue iron distribution [9,10].
Electroacupuncture (EA) is another type of acupuncture that originated from the combination of acupuncture and electrical stimulation. Because of its relatively simple, inexpensive, and highly feasible treatment characteristics, it is generally accepted by stroke patients in clinical practice [11]. Our previous study [12] found that acupuncture improved the symptoms of neurological deficits and reduced pathological inflammatory injury in rats with cerebral hemorrhage by regulating the expression of HO-1 and inflammatory factors; however, there has been no in-depth study from the perspective of iron metabolism. In this experiment, we used electroacupuncture at the Baihui and Dazhui acupoints to treat rats with cerebral hemorrhage to observe the effect of electroacupuncture on the expression of Hepc, ferritin(Ft), transferrin(Tf), and transferrin receptor(TfR) proteins in the surrounding tissue of the hematoma. Introducing siRNA to interfere with the expression of Hepc, by constructing siRNA against the Hepc gene, the gene expression of Hepc was reduced, so as to explore the correlation between brain iron metabolism and Hepc expression after cerebral hemorrhage and the effect of electroacupuncture to explore the pathological mechanism of secondary brain injury after ICH from the perspective of iron metabolism.

Materials and Methods
2.1. Experimental Animals. 90 healthy 8-week-old SPF-grade male Wistar rats weighing 300 ± 20 g (provided by the Animal Experiment Center, Norman Bethune Health Science Center of Jilin University, Animal License No. SCXK(Ji) 013-0004) were housed in a ventilated room in the Brain Function and Neurorehabilitation Laboratory of Heilongjiang University of Chinese Medicine, maintained at a temperature of 22 ± 3°C, relative humidity of 60 ± 5%, and 12 h/12 h alternating light/dark cycles, and there is an ultraviolet disinfection system to disinfect the animal room on a regular basis, the animal room is well ventilated, and the food and water sources are sufficient and hygienic. The experiment was approved by the Animal Care and Use Committee at the Heilongjiang University of Chinese Medicine (No. hljngnsysll202108), and experiments followed the relevant provisions of the "Guideline on Welfare of Laboratory Animals" issued by the Ministry of Science and Technology.

Model Construction.
A rat model of cerebral hemorrhage was constructed by referencing the literature [13,14]. The rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), fixed in the prone position on the stereotaxic apparatus, and prepared for skin disinfection. A midline incision was made, and a periosteal dissector was used to dissect the periosteum and expose the fontanelle and coronal suture; 3.5 mm to the right and 0.2 mm posterior of the fontanelle point (Bregma's point) was located and a circular hole of 1.0 mm in diameter was made with a dental drill to reach the dura surface. The rat's tail was disinfected using alcohol, and the tail was cut 3 cm from the caudal end. Fifty microliters of blood was obtained using a microinjector, which was fixed on the stereotaxic apparatus, and the needle was inserted approximately 6 mm along the drill hole. Fifty microliters of nonheparinized blood was pushed into the caudate nucleus at a rate of 20 μl/min, and the needle was left in place for approximately 5 min and slowly withdrawn. During needle retention, alcohol-soaked cotton balls were used to bandage the wound at the cut end of the rat's tail, gentamicin was sprayed locally after surgery, the skull wound was closed with dental cement, the scalp was sutured, and the local skin was disinfected with Iodine phenol. Animals in the sham group underwent the same surgical procedures as those in the model group, but no blood injection was performed.
Six hours after surgery was performed, the Berderson scoring method [15] was used to determine successful animal models. Rats with scores of 1 to 3 were included in the experiment. We used hematoxylin-eosin staining for pathological detection of cerebral ischemia model.

Grouping and Intervention
Methods. Seventy-two male Wistar rats were divided into sham, model, EA group, Hepc siRNA group, EA+Hepc siRNA, or control siRNA groups using a random number table method, with 12 rats in each group, as shown in (Figure 1). The remaining 18 rats will be used as substitutes in the event of an accident in the experiment.
Animals in the sham group received various surgical procedures similar to those performed in the treated group, but without blood injection. Normal handling and fixation were performed without any intervention. Animals in the model group only underwent cerebral hemorrhage modeling; normal handling and fixation were performed without any intervention.
In EA group, a cerebral hemorrhage model was induced. Treatment was started on day 1 after surgery until the animals were euthanized. By referring to the "Development of Acupoint Atlas of Rats" [16], the Baihui acupoint (in the middle of the parietal bone) and Dazhui acupoint (between the seventh cervical vertebra and first thoracic vertebra in the middle of the back) were selected, and the Huatuo no. 30 0.5-inch milli-needle was used to perform acupuncture at the Baihui and Dazhui acupoints, at a depth of 10 mm and connected to a G6805-2A electroacupuncture instrument (Shanghai Huayi). Stimulation was provided with sparse and dense waves, at a frequency of 2 Hz/15 Hz, intensity of 2 mA, and duration of 30 min. Acupuncture was performed once per day.
In the Hepc siRNA group, a viral suspension of 5 μl Hepc siRNA was injected into the lateral ventricle 30 min before 2 Mediators of Inflammation induction of a cerebral hemorrhage model. Normal handling and fixation were performed without any intervention.
In the EA+Hepc siRNA group, 5 μl of viral suspension of Hepc siRNA was injected into the lateral ventricle 30 min before induction of a cerebral hemorrhage model. The remaining steps were the same as those in the EA group.
In the control siRNA group, a viral suspension of 5 μl scrambled siRNA was injected into the lateral ventricle 30 min before model construction to construct a model of cerebral hemorrhage. Normal handling and fixation were performed without any intervention.

Synthesis of Hepc siRNA and Lentiviral Vector
Construction. A small interfering RNA fragment of Hepc (Hepc siRNA) and an ineffective small interfering RNA fragment as a control (scramble siRNA) were designed and synthesized by Wanlei Life Sciences (Shenyang, China) biological company. Plasmids were constructed and packaged with lentiviral particles and stored briefly at 4°C for subsequent experiments.

Observation Markers and Testing
Methods. After surgery (3 d, 7 d, and 14 d), neurological deficit scoring was carried out for each group of rats. At 14 days after surgery, rats were deeply anesthetized using intraperitoneal injection of 2% pentobarbital sodium (0.4-0.5 ml/100 g). Six rats were selected from each group for intracardiac perfusion. Brain tissue was then excised from rats in each group to remove the olfactory bulb and tissue from 4 mm in front of the frontal pole, from the middle of the right cerebral hemisphere, with the injection needle tract as the center, and each sample was sliced with a coronal thickness of about 5 mm. Tissues were embedded in paraffin and stored for immunohistochemistry; six other rats were selected, brain tissue was then excised from rats in each group to remove the olfactory bulb and tissue from 4 mm in front of the frontal pole, from the middle of the right cerebral hemisphere, with the injection needle tract as the center, and each sample was sliced with a coronal thickness of about 5 mm, frozen at −80°C, and kept for subsequent total iron content assay, western blotting, and reverse transcription polymerase chain reaction (RT-PCR) assay.

Determination of Total Iron Content in Perihematoma
Tissue. A 0.05 g sample of brain tissue was accurately weighed, rinsed repeatedly with deionized water, dried in a 60°C oven, nitrated with high-quality pure nitric and perchloric acids, and underwent high temperature ashing until colorless or slightly yellowish transparent crystals were obtained. Its ionized water was added to 5 ml, and the total iron content in brain tissue was measured using flame atomic absorption spectrometry [19].
2.9. Detection of Hepc, Ft, Tf, and TfR Expressions in Perihematomal Tissue Using Immunohistochemistry. The expression of Hepc, Ft, Tf, and TfR in the perihematomal tissue was detected through brain tissue embedding, sectioning, clearing and hydration, antigen retrieval, hydrogen peroxide incubation, goat serum blocking, primary antibody incubation, secondary antibody incubation, horseradish enzyme labeling, DAB color development, hematoxylin counterstaining, dehydration, clearing, mounting, and microscopic examination. The mean optical densities of proteins were measured.  After total protein extraction, the protein concentration was determined using the BCA method, followed by resolution with SDS-PAGE at the corresponding concentration, transfer to membranes, addition of the corresponding primary and secondary antibodies for immunoreactivity, and addition of ECL luminescent solution for exposure. The relative expression of the target protein and β-actin was calculated by detecting the grayscale value (OD) of the bands using Quantity One analysis software (BIO-RAD company USA).
2.11. RT-PCR Measurement of Interference Efficiency of Hepc siRNA in Perihematoma Tissues. Rat brain tissues were collected, and total RNA was extracted by adding TRIzol. cDNA was obtained through reverse transcription of total RNA and used as the sample for detection of the β-actin and target genes. A housekeeping gene was used for normalization, and the primers shown in Table 1 were provided by Wanlei Life Sciences Co., Ltd. (Shenyang, China). Relative expression analysis of the above markers was performed using the 2-ΔΔCt method.
2.12. Statistical Processing. The data were statistically analyzed using SPSS22.0. Normally distributed quantitative data are expressed as mean ± standard deviation ( x ± s), and oneway analysis of variance was used to compare between groups at each time point. The least significant difference t -test was used for pairwise comparison. A difference with P < 0:05 was considered statistically significant.  Figure 2(a). All rats had an mNSS score of 0 before scoring. The sham group had no significant postoperative neurological deficits. The rats in the model group showed significant signs of neurological deficit by day 3 and still showed signs of neurological deficit on days 7 and 14. Compared with the model group, the mNSS scores of rats in the electroacupuncture group were significantly lower at all time points (P < 0:01). Compared with the EA group, the mNSS scores of rats in the Hepc siRNA and EA+Hepc siRNA groups were significantly lower at all time points (P < 0:01). Compared with the Hepc siRNA group, the neurological function scores of rats in the EA+Hepc siRNA group were significantly lower at all time points (P < 0:01) (Figure 2(b)).

Comparison of Total Iron Content of Rats in Each Group.
Rats in the sham group had a small amount of iron in the brain after surgery. The iron content in the brains of rats in the model group was significantly higher on days 3, 7, and 14. Compared with the model group, the iron content in the brains of rats in the EA group was significantly lower at all time points (P < 0:01). Compared with the electroacupuncture group, the iron content in the brains of rats in the Hepc siRNA and EA+Hepc siRNA groups at all time points was significantly lower (P < 0:01). Compared with the Hepc siRNA group, the EA+Hepc siRNA group had significantly lower iron content in rat brain at all time points (P < 0:01) (Figure 2(c)).
3.3. Immunohistochemical Results of Hepc, Ft, Tf, and TfR in Perihematoma Tissue. On day 14 after surgery, weak positive expression of Hepc, Ft, Tf, and TfR was seen in the sham group. Expression of Hepc, Ft, Tf, and TfR protein was significantly higher in the model group (P < 0:01). Compared with that in the model group, Hepc, Ft, Tf, and TfR protein expression was lower in the EA group   Mediators of Inflammation (P < 0:01). Compared with that in the EA group, Hepc, Ft, Tf, and TfR protein expression was lower in the Hepc siRNA and EA+Hepc siRNA groups (P < 0:01). Compared with that in the Hepc siRNA group, Hepc, Ft, Tf, and TfR protein expression was lower in the EA+Hepc siRNA group (P < 0:01) (Figure 3).

Western Blotting Results of Hepc Protein in
Perihematoma Tissue. On day 14 after surgery, weak positive expression of Hepc was seen in the sham group. Hepc protein expression was significantly higher in the model group (P < 0:01). Compared with that in the model group, Hepc protein expression was lower in the EA group (P < 0:01). Compared with that in the EA group, Hepc protein expression was lower in the Hepc siRNA and EA+Hepc siRNA groups (P < 0:01). Compared with that in the Hepc siRNA group, Hepc protein expression was lower in the EA+Hepc siRNA group (P < 0:01) (Figure 4).  Hepc mRNA expression was significantly higher in the model group (P < 0:01). Compared with that in the model group, Hepc mRNA expression was lower in the EA group (P < 0:01). Compared with that in the EA group, Hepc mRNA expression was lower in the Hepc siRNA and EA +Hepc siRNA groups (P < 0:01). Compared with that in the Hepc siRNA group, Hepc mRNA expression was lower in the EA+Hepc siRNA group (P < 0:01) ( Figure 5).

Discussion
Neurological deterioration in patients with cerebral hemorrhage is attributed to several modifiable factors, including acute hematoma growth, intracerebroventricular dilatation, and perihematoma edema [20]. After cerebral hemorrhage, iron ions released by erythrocyte lysis in the hematoma play a key role in mediating secondary neuronal injury and edema formation [4,21]. Iron levels in brain tissue surrounding the lesion remain significantly higher than normal during recovery, and the deposited iron can be reactivated to cause delayed brain injury, although the pathogenesis is unclear [22,23]. Hemoglobin released by the disintegration of red blood cells in the blood can be decomposed in the presence of HO-1 to produce Fe 2+ , CO, and biliverdin, resulting in iron overload in the brain, where the nonheme iron content can increase by threefold; the main mechanisms of neurodegeneration in hemorrhagic stroke are iron and heme-induced ROS production, amplification of inflammatory responses, direct toxic effects of iron and heme, and glutamate-induced excitotoxicity [24,25]. As a result, damaged mitochondria produce many reactive oxygen species [26]. The resulting disruption of glutamine metabolism leads to dysfunction of antioxidant pathways and hydroxyl radical synthesis, which in turn leads to neuronal ferroptosis [27][28][29]. Iron ions are involved in the metabolic processes of brain cells, and low or high levels of iron ions in brain tis-sue can cause functional brain damage [7]. The amount of iron deposition in the brain tissue of rats after cerebral hemorrhage gradually increases in the brain injury area, pathological damage of brain tissue gradually increases, and the amount of iron deposition positively correlates with the neurological severity score of rats, suggesting that excessive iron deposition in brain tissue is an important factor leading to brain injury [30]. The imbalance of iron metabolism in patients after cerebral hemorrhage and elevated serum iron-regulating factor levels are associated with poorer prognosis in patients, and the trend of elevation is consistent with the severity of neurological deficit and prognosis of patients [31].
After cerebral hemorrhage, iron metabolism in the brain is imbalanced and the body activates the Fe-transferrin -TfR pathway to maintain its own homeostasis, promoting the efflux of iron ions while converting them into Ft for storage. When excess free iron exceeds the saturation capacity of Tf in the brain and generates large amounts of oxygen radicals through the Fenton reaction, it induces inflammatory  Mediators of Inflammation responses and apoptosis [32,33]. Wang et al. [21] demonstrated that iron overload after ICH caused oxidative damage to brain tissue, damage to the blood-brain barrier, microglial activation, and production of large amounts of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which aggravate brain edema and neuronal death. The aforementioned studies suggest that ameliorating the inflammatory response induced by the imbalance of iron metabolism in the brain is one of the key targets for the treatment of cerebral hemorrhage. Most evidence suggests that Tf/TfR1 pathway is the main route of iron transport through the luminal membrane of capillary endothelial cells [34,35], possibly via the ferroportin1/hephaestin and/or fer-roportin1/ceruloplasmin export system to transport iron as Fe 2+ across the isolated membrane into the brain parenchyma [36][37][38]. Therefore, how to effectively regulate iron deposition around the hematoma in patients with cerebral hemorrhage has been a hot topic in cerebral hemorrhage research in recent years. Hepc is a newly discovered important ironregulating hormone, which is a key substance in regulating iron homeostasis and iron metabolism in human body and plays an important role in maintaining the balance of iron metabolism in the body and cells, including hereditary hemochromatosis protein, Tfr2, and hemojuvelin [39]. The inflammatory response, hypoxic activity, erythropoietic activity, and changes in iron level that occur in the body of patients after cerebral hemorrhage lead to an increase in serum Hepc levels [40]. Hepc plays an important role in the clinical treatment of patients with cerebral hemorrhage by maintaining the balance of iron metabolism in the body through the regulation of serum iron concentration, the regulation of intestinal iron absorption, and the regulation of tissue iron distribution [41]. Clinical studies have found that serum Hepc was higher in patients with cerebral hemorrhage with poor prognosis at 1, 3, 5, and 7 d than in patients in the good prognosis group (P < 0:05), suggesting that serum Hepc levels in patients after cerebral hemorrhage are related to the prognostic outcome of patients [42]. Current studies on Hepc have shown different results, which may be related to the differential expression of Hepc in different parts of the brain [43]. At the same time, the production of Hepc is regulated by iron levels. In pathological states, increased circulating iron leads to upregulation of Hepc expression through feedback regulatory mechanisms, and Hepc forms complexes with membrane iron transport proteins, which induce ubiquitination of membrane iron transport proteins and negatively regulate the reduction of intracellular iron output [44,45]. Therefore, Hepc, as a key factor of iron metabolism, plays an important regulatory role in many ways and is a new target and prognostic marker for the treatment of cerebral hemorrhage in clinical settings. Based on the above studies, in the present study, we investigated the changes in Hepc, Tf, TfR, and Ft in order to explore the mechanism of cerebral hemorrhage cases from the perspective of iron metabolism.
In the book "Compendium of Acupuncture and Moxibustion" by Yang Jizhou of the Ming Dynasty, it is stated that a three-edged needle should be used for those with acute stroke and 12 well points should be used for acupuncture. For slanted mouth and eyes, numbness of the hands and feet, and confusion, the Dazhui, Fengchi, Quchi, Zusanli, and Baihui acupoints should be used in acupuncture. For yin syndrome stroke, supplementation should be performed first followed by dissipation, with acupuncture performed on the contralateral side first and then the ipsilateral side. Among the above acupoints, Baihui is the Du meridian, which is located at the intersection of the line connecting the tips of the ears and the midline of the top of the head, where the three yang and five meetings are located, and the depth of which is where the brain is located. The Dazhui point is located in the depression below the spinous process of the 7th cervical vertebra and is where the Yang-heat Qi from the three Yang of the hands and feet converges into the Du meridian. This allows the Yang-heat qi to travel up through this point to the top of the head along the Du meridian. It can be seen that acupuncture at Baihui and Dazhui points can internally open the Du meridian and externally; they can flow through the three yang [46]. Therefore, Baihui and Dazhui points are the key points for stroke and improvement of brain function [47]. Dong et al. [48] used head acupuncture to treat the acute phase of cerebral hemorrhage, selecting head acupoints and applied acupuncture to the area from Baihui to the Taiyang acupoint to treat acute cerebral hemorrhage, breaking the paradigm that head acupuncture is prohibited in acute cerebral hemorrhagic stroke. So the Baihui and Dazhui acupoints were chosen to treat ICH rats in this study. Acupuncture can effectively promote the recovery of limb function and activities of daily living in patients with cerebral hemorrhage, and the earlier the acupuncture intervention is provided, the better the outcome [49,50]. Electroacupuncture is a therapy in which a very slow current close to that of natural human bioelectricity is passed through the needles to treat a disease, which can cause excitation in corresponding cortical areas, enhance brain electrical activity, stimulate sensory nerve impulses, induce the corresponding part of brain cells to awaken, and restore brain function. Several studies have shown that electroacupuncture can reduce the volume of cerebral edema in patients; improve their neurological, vascular, and cognitive abilities; and improve their activities of daily living and quality of life [51][52][53][54][55]. And studies have confirmed that acupuncture can improve neuronal ferroptosis after cerebral hemorrhage, thereby reducing the degree of nerve damage [56,57].
In this experiment, the mNSS score results revealed that the model group rats showed different degrees of neurological deficit. The neurological scores of rats in the model group rats were highest on day 3 after surgery, and the neurological deficit symptoms of the rats gradually decreased from day 7 after surgery. Some neurological deficits were still present on day 14 after surgery, suggesting that the cerebral hemorrhage model was successful with neurological deficit symptoms. On days 3, 7, and 14, the neurological function scores were significantly lower in the electroacupuncture group than in the model group (P < 0:01), indicating that electroacupuncture could effectively reduce neurological function scores in rats with cerebral hemorrhage. At the same time points, the neurological function scores of the Mediators of Inflammation Hepc siRNA group were lower compared than those of the model group and electroacupuncture groups (P < 0:01), indicating that silencing the Hepc gene could also reduce neurological deficits in rats with cerebral hemorrhage. The EA+Hepc siRNA group had the lowest neurological function score and the most significant improvement in neurological deficit relative to the model, EA, and Hepc siRNA groups (P < 0:01). This shows that electroacupuncture at the Baihui and Dazhui acupoints can effectively reduce the neurological function score and improve the symptoms of neurological deficits in rats with cerebral hemorrhage. The total iron content in the brain showed the same trend as the neurological score, suggesting that the total iron content in the brain of rats with hemorrhage was directly proportional to the neurological deficit degree (P < 0:01) The immunohistochemistry, western blot, and RT-PCR results also reflected that electroacupuncture decreased Hepc protein and gene expression and decreased Ft, Tf, and TfR protein expressions (P < 0:01).
After cerebral hemorrhage rats have increased intracerebral iron content and obvious symptoms of neurological deficits, we hypothesized that electroacupuncture may promote intracerebral iron metabolism and reduce symptoms of neurological deficits in rats with cerebral hemorrhage by decreasing Hepc protein and gene expression and decreasing Ft, Tf, and TfR protein expressions. However, this study also has some limitations; although we confirmed the therapeutic effect of electroacupuncture in intracerebral iron metabolism after cerebral hemorrhage, we only based on animal experiments and could not cover the disease of the situation, so the results of this study should be used in the context of the specific situation; in addition, we only studied the intracerebral iron metabolism in the acute phase of cerebral hemorrhage and did not explore the hyperacute phase. Future research directions will include exploring the limitations of this study and conducting a more in-depth study of its mechanism of action.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.